The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). Upon ligand binding, EGFR stimulates downstream cell signaling cascades that influence cell proliferation, migration, apoptosis, and survival. The two FDA-approved anti-EGFR antibodies, chimeric monoclonal antibody (mAb) Cetuximab (Erbitux™) and fully human mAb Panitumumab (Vectibix™), are high-affinity mAbs with therapeutic applications in cancers of the colon and head & neck (Wheeler et al. 2010, Nat Rev Clin Oncol. 7, 493-507). Due to the higher specific and affinity as comprised with peptide ligand or small molecular, antibodies not only used for therapy but also used as vectors to deliver drugs into the target cells.
Despite the success of IgG, such as Cetuximab and Panitumumab, used in clinical trials, a large size of IgG (˜150 kDa) may affect the tumor penetration (Jain et al. 2005, Cancer Res. 65, 7840-6). Many kinds of antibody formats, such as single chain variable fragments (scFvs) and scFv fused with Fc domain (scFv-Fc), have been engineered to improve tumor penetration. The naturally devoid of light chain antibodies of camels and llamas provide new perspectives in the antibody engineering area (Hamers-Casterman et al. 1993, Nature, 363, 446-8.). Single-domain antibodies (sdAbs, also called dAbs) are the smallest antigen binding fragment based on a single variable domain of human antibodies and are highly soluble when expressed in a recombinant system (Jespers et al. 2004, Nat Biotechnol. 22, 1161-5.). Recently, a reference protocol for phage display selection of human antibody fragment via filamentous bacteriophage was set up (Lee et al. 2007 Nat Protoc. 2, 3001-8.). For enhancing the hit rates, the phage display selection based on antigen expressing cells, to mimic the physiological form of receptors, has also been reported in the literature (Heitner et al. 2001 J Immunol Methods. 248, 17-30). The EGFR-transfected 32D (32D-EGFR) cell line was reported as a suitable EGFR pathway inhibitor screening system based on interleukin-3 (IL-3) and EOF dependent cell survival (Lin et al. 2008, Anal Biochem. 377, 89-94).